Eur J Nucl Med Mol Imaging (2011) 38:81–89
DOI 10.1007/s00259-010-1613-z

ORIGINAL ARTICLE

Early response assessment in prostate carcinoma
by 18F-fluorothymidine following anticancer therapy
with docetaxel using preclinical tumour models
Nobuyuki Oyama & Yoko Hasegawa & Yasushi Kiyono & Masato Kobayashi &Yasuhisa Fujibayashi & Datta E. Ponde & Carmen Dence & Michael J. Welch &Osamu Yokoyama

Received: 13 June 2010 / Accepted: 25 August 2010 / Published online: 29 September 2010
# Springer-Verlag 2010

Abstract
Purpose The aim of the study was to assess the potential
usefulness of 3-deoxy-3-18F-fluorothymidine (FLT) as aradiopharmaceutical for imaging the early therapeutic
effects of docetaxel (DTX) on tumour proliferation inhormone-refractory prostate cancer (HRPC).
Methods Cells of the androgen-independent human prostate
tumour cell line, 22Rv1, were implanted in athymic malemice. Approximately 3 weeks after cell implantation, themice were treated with DTX or vehicle. Before and after
the treatment, the mice were imaged with a microPETFocus-F120 scanner (Concorde Microsystems, Knoxville,
TN, USA) using FLT and 18F-fluorodeoxyglucose (FDG).
Tracer accumulations in the tumours were then analysedand compared with the proliferation activity and apoptotic
index of the tumours. In a separate cell study, 22Rv1 cells

N. Oyama (*) : Y. Hasegawa : O. Yokoyama
Department of Urology, Faculty of Medical Sciences,
University of Fukui,23-3 Matsuoka-Shimoaizuki,
Fukui 9101193, Japane-mail: urono@u-fukui.ac.jp
Y. Kiyono : M. Kobayashi : Y. Fujibayashi
Biomedical Imaging Research Center, University of Fukui,
Fukui, JapanD. E. Ponde : C. Dence : M. J. Welch
Mallinckrodt Institute of Radiology,
Washington University School of Medicine,
St. Louis, MO, USA
M. J. Welch
Siteman Cancer Center,
Washington University School of Medicine,
St. Louis, MO, USA

were treated with DTX, then incubated with FLT or FDG
and examined for their tracer uptake.
Results The microPET imaging showed a significant
decrease of FLT uptake in tumours after administration of
DTX, while the changes of FDG uptake were minimal.
Immunohistochemical analysis of the tumours revealed thatthe changes of FLT uptake were well correlated with thoseof proliferation activity but not with the apoptotic index. In
vitro studies demonstrated that the significant decrease of
FLT uptake in the cells after incubation with DTX
correlated with the % S-phase cell fraction, while there
were only minimal changes in the prostate-specific antigen
concentration of the cell medium and FDG uptake in thecells.
Conclusion These results indicate that FLT is a promising
tracer for monitoring the early effects of anticancer therapy
with DTX in patients with HRPC.
Keywords PET . Prostate cancer . Docetaxel .
Tumour proliferation . 18F-Fluorothymidine

Introduction
Prostate cancer is the most commonly diagnosed cancer andis the second leading cause of cancer death in men over theage of 40 years in the USA [1]. Androgen dependency is acharacteristic behaviour for most of the prostate cancers at
first presentation [2]. Therefore, androgen ablation therapy(AAT) has been employed as the first-line treatment forpatients with locally advanced disease or metastatic disease.
Nonetheless, relapse after AAT remains a major problem.
Many of the patients with hormone-refractory prostate cancer(HRPC) receiving AAT relapse within a few years of

82

treatment, and although chemotherapy is often considered oneof the options for treating relapsed cancers, it is not as effective for prostate cancer as for other types of malignancy [3].
Recently, docetaxel (DTX)-based chemotherapy has
been shown to improve survival in this disease [4]. Afterinitiation of DTX therapy, conventional imaging modalities
such as ultrasonography, computed tomography (CT) andmagnetic resonance imaging (MRI) are employed tomonitor the therapeutic effect by evaluating the anatomicalchanges of cancer lesions; however, the usefulness of thesemodalities is limited because they often require several
months of treatment for the first accurate assessment. The
anticancer effects of DTX can also be monitored bymeasuring the levels of serum prostate-specific antigen(PSA), but the reliability of this method is limited because
HRPC tumours may have lost the ability to produce PSA or
may produce less PSA than androgen-dependent prostatecancer tumours. Thus, there is a need for a more reliablediagnostic marker for the evaluation of the early therapeutic
effect of DTX in patients with HRPC.
Positron emission tomography (PET) using the radiopharmaceutical 2-[18F]fluoro-2-deoxy-D-glucose (FDG) has
been widely accepted as a highly effective means forimaging a wide variety of cancers. FDG PET has been
proven useful for monitoring the efficacy of prostate cancer
treatments in both animal models [5] and humans [6, 7]. In
these previous studies, treatment efficacy was monitored byusing FDG PET to visualize metabolic changes of glucose
in the prostate tumours following treatment. However,
because the tumour grows relatively slowly in most cases,
FDG PET has been previously shown not to be appropriate
for initial diagnosis of primary as well as distant disease of
prostate cancer [8, 9]. Urine excretion of this tracer isanother disadvantage for imaging of prostate cancer andother urological malignancies.
Positron-labelled choline or acetate analogues have beenreported to have higher sensitivity for the tumour detectionof prostate cancer [10–12]. However, the usefulness ofthese tracers as cell proliferating markers of the tumours islimited [5, 13, 14], which may not be suitable for theevaluation of the early effect of anticancer therapy.
18
F-3′-Deoxy-3′-fluorothymidine (FLT) has been introduced as a radiopharmaceutical for use in assessing tumour
proliferation by PET imaging [15]. Evaluation of thetumour proliferative activity by PET using FLT may havepotential for assessing the viability of the tumour as well as
the early effects of cancer therapy. In recent clinical studies,
FLT uptake was higher in lung cancer tissue than in benign
masses, indicating its usefulness for the differential diagnosis between malignant and benign tumours [16, 17].
Another previous study reported the use of FLT PET toevaluate breast cancer treatments [18]; the results indicatedthat FLT PET after the first course of chemotherapy was

Eur J Nucl Med Mol Imaging (2011) 38:81–89

useful for predicting the long-term efficacy of chemotherapy regimens in women with breast cancer.
In our previous study, we demonstrated that FLT uptake
in prostate tumours declined after initiation of AAT in an
animal model and that this decline was correlated with thedecrease of tumour size [5]. This was the first study whichshowed the possibility of using FLT PET imaging tomonitor the therapeutic effects of AAT on prostate cancer.
The purposes of the present study were to determine
whether microPET with FLT is useful for monitoring theearly therapeutic effects of DTX in vitro and in vivo andalso to determine whether FLT uptake in tumours correlateswith the actual proliferative activity after DTX therapy.

Materials and methods
Radiochemical synthesis
FLT was synthesized as described previously. In brief, 18Ffluoride was produced via the 18O(p, n)18F nuclear reaction
by irradiating isotopically enriched 18O-water with 15–
16 MeV protons using either the Washington University
Cyclotron Corporation CS-15 or the Japan Steel Works(JSW) BC-16/8 medical cyclotron. The radioactivity
emerging from the target was resin treated to reclaim the18
O-water. 18F-Fluoride was eluted from the resin in a solution of 0.02 N potassium carbonate and used in subsequent
reactions. FLT was synthesized starting with anhydrothymidine and microwave-mediated nucleophilic displacement byfluoride ion followed by acidic hydrolysis gave FLT in just60 min [19]. The radiochemical yield was 12±4% (decay
corrected) and the radiochemical purity was >99%.
Cellular uptake study
A well-established 22Rv1 human androgen-independent
prostate adenocarcinoma cell line (American Type Culture
Collection, Manassas, VA, USA) was chosen for ourcellular uptake study. DTX was gifted by Sanofi-Aventis(Paris, France). Cells were maintained in RPMI 1460
medium (Gibco by Invitrogen, Carlsbad, CA, USA)
containing 10% fetal bovine serum (FBS, Gibco by
Invitrogen, Carlsbad, CA, USA) at 37°C in an atmosphereof 95% air and 5% CO2. The cells were cultured in 24-wellplates in quintuple at a concentration of 2×105 cells per
well. Forty-eight hours after incubation, when the cells
reached 50% confluence in the wells, they were divided
into three groups: group 1 was supplemented with 10%
FBS medium, group 2 with 10% FBS medium with 10 nM
DTX, and group 3 with 10% FBS medium with 100 nM
DTX. After 24, 48 and 72 h of incubation with therespective media, 0.37 MBq of FLT or 0.37 MBq of FDG

Eur J Nucl Med Mol Imaging (2011) 38:81–89

was added to each well and the cells were further incubatedfor 2 h. After incubation with the tracers, 22Rv1 cells were
harvested and the number of cells, % uptake of radioactivity
into the cells, PSA concentration in the media and % S-phasefractions of cells in each group were determined.
Flow cytometric method
Immediately following the drug treatments describedabove, cells were harvested by trypsinization to obtain asingle cell suspension. The cells were sedimented bycentrifugation and treated with a CycleTEST™ PLUS
DNA Reagent Kit (Becton Dickinson, San Jose, CA,
USA) to prepare samples for DNA analysis on a flow
cytometer, according to the manual from the manufacturer.
The cell profiles of samples were analysed by flow
cytometry (FACSCalibur, Becton Dickinson, San Jose,
CA, USA). The resulting DNA histograms were analysed
for determination of the % S-phase fraction using ModFit
LT software (Becton Dickinson, San Jose, CA, USA).
MicroPET imaging
Animal experiments were conducted in compliance with the
Guidelines for the Care and Use of Research Animals
established by the Animal Studies Committee at our institution. Four- to six-week-old athymic nu/nu male mice wereobtained from Charles River Laboratories (Wilmington, MA,
USA). 22Rv1 cells have wild-type androgen receptors, growin an androgen-independent fashion and produce PSA. Cells(2×105) were implanted subcutaneously into the right flankof the mice. Three weeks after tumour implantation, the mice
were imaged in the prone position in a microPET-FocusF120 scanner (Concorde Microsystems, Knoxville, TN,
USA). For the baseline PET scans, mice were firstanaesthetized with 1–2% isoflurane and placed in a prone
position in a custom-designed holder that allows simultaneous imaging of two mice. The entire holder was placednear the centre field of view (CFOV) of the microPET
scanner. The mice were injected intravenously with 7.7 MBq
of 18F-FLT and a 10-min static scan was obtained at 2 h
post-18F-FLT injection. A 10-min static scan was also
obtained at 1 h post-18F-FDG injection (7.7 MBq) on thenext day. Following baseline PET imaging, six mice weretreated with intravenous DTX (25 mg/kg) weekly for
2 weeks. Six control mice received no treatment. One week
after the second DTX treatment, microPET imaging was
repeated with the same protocol as described for the baseline
studies. The methods of data collection and the reconstruction for microPET imaging have been described previously[5]. Briefly, all raw data were first sorted into threedimensional (3-D) sinograms, followed by Fourier rebinningand 2-D filtered backprojection (FBP) reconstruction using a83

ramp filter cutoff at the Nyquist frequency. A region ofinterest (ROI) was placed on each tumour or other organs in
the transaxial microPET images that included the entire
tumour or organ volume. The average radioactivity concentration within a tumour or an organ was obtained from theaverage pixel values within the multiple ROI volume. To
eliminate the dependency on the injected total amount ofradioactivity, tumour to muscle ratios were used to compare
the FLT uptake in the tumour before and after the treatment.
In these microPET scans, the attenuation corrections were
not applied, since the accuracy of the measured attenuation
correction was poor with this scanner, and since the amountof attenuation from the mouse body was relatively small andthe shape of the mouse body did not change significantly
among subjects; instead, the attenuation correction factors
were incorporated into the system calibration. With theimage reconstruction algorithm and filter described above,
the resolution of this microPET system is between 2.0 and
3.0 mm full-width at half-maximum (FWHM) in each of thethree dimensions within a 5-cm imaging FOV around thecentral axis of the tomograph. The partial volume effect inestimating the ROI values from microPET images is small inthese studies, since the implanted tumours were more than
1 cm in diameter, which was well above the resolution limitof this system [20]. To assess the PSA production of
implanted tumour, the serum PSA values of mice were also
determined after the treatment.
Immunohistochemical examination of the 22Rv1 tumours
For histological evaluation of the degree of tumour
proliferation and apoptosis, the tumours were excised, fixedin formalin, embedded in paraffin and cut into sections.
Tumour sections were stained with mouse monoclonal
antibodies for proliferating cell nuclear antigen (PCNA)(Dako, Carpinteria, CA, USA). PCNA labelling is anestablished measure of proliferation. PCNA is a 36,000
molecular weight, acidic, non-histone, nuclear protein inwhich the expression is associated with the late G and S
phase of the cell cycle. A PCNA assay was performed on
tumour slices, and proliferating cells were detected. Eachexperiment included three or four slices for each treatment.
For each slice, a total of 100–200 nuclei were counted. For
immunohistochemical detection of apoptosis, a TUNEL
assay was performed using an ApopTag Peroxidase Kit(Intergen, Purchase, NY, USA). The TUNEL assay was
performed on tumour slices obtained from mice after
microPET scanning. Apoptotic nuclei were expressed as
the percentage of the total nuclei counted. The tumour
slices were fixed overnight in 10% neutral bufferedformalin. After standard processing for the preparation ofsections from paraffin-embedded tissues, the apoptotic
tumour nuclei were detected using the TUNEL assay. Each

84

Eur J Nucl Med Mol Imaging (2011) 38:81–89

experiment included three or four slices for each treatment.
For each slice, a total of 100–200 nuclei were counted.
Statistical evaluation
All data are reported as the mean ± the sample standard
deviation (SD). Analysis of variance (ANOVA) was used tocompare the tracer uptake, number of cells, apoptosis
fraction and cell cycle distribution. An unpaired t test wasused to compare the tumour growth rate, serum PSA value,
apoptotic cell rate and PCNA-positive cell rate. Statisticalsignificance was established at p<0.05.

Results
Cellular uptake study
Figure 1a shows the number of 22Rv1 cells in each group.
After 24-h incubation, there was no significant difference inthe number of 22Rv1 cells among the standard medium andNumber of cells (1x105)

a

70

0nm

60

10nm

50

100nm

40
30
20

**
***

10
0
24
4

***

**

4
48

72

Incubation time (h)Medium PSA value (ng/mL)

b

4
3.5
3

0nm
10nm
100nm

the media with 10 nM or 100 nM DTX (standard vs 10 nM
DTX vs 100 nM DTX after 24-h incubation: 10.49±5.17×
105 vs 8.00±2.87×105 vs 5.07±1.47×105). Following
continuous incubation with the media containing DTX,
the number of cells appeared to be lower than that instandard medium (standard vs 10 nM DTX or 100 nM DTX
after 48-h incubation: 28.62±11.17×105 vs 9.33±2.57×
105, p<0.01 or 6.31±2.51×105, p<0.01; after 72-h incubation: 44.09±14.89×105 vs 11.38±4.99×105, p<0.01 or
4.89±0.99×105, p<0.01). Figure 1b shows the PSA concentration in the media for each group. After 24-h incubation,
there was a small difference in the PSA concentration among
the three groups (standard vs 10 nM DTX vs 100 nM DTX at
24 h of incubation: 0.77±0.05 ng/ml vs 0.580±0.03 ng/ml vs
0.55±0.04 ng/ml). Following continuous incubation with themedia containing DTX, the PSA concentration appeared to
be lower than in the standard media (standard vs 10 nM DTX
or 100 nM DTX after 48-h incubation: 1.92±0.06 ng/ml vs0.95±0.05 ng/ml, p<0.001 or 0.87±0.05 ng/ml, p<0.001;
after 72-h incubation: 3.07±0.28 ng/ml vs 1.21±0.11 ng/ml,
p<0.001 or 0.94±0.07 ng/ml, p<0.001). As shown in
Fig. 2, after incubation in each medium, the FDG or FLT
uptake in the cells was determined. There was no
statistically significant difference in FDG uptake in the cells
after 24-h incubation among the three groups. Following
continuous incubation with the media containing DTX, theFDG uptake in the cells appeared to be lower than in thestandard media (standard vs 10 nM DTX or 100 nM DTX
after 48-h incubation: 2.81±0.53% uptake/1×105 cells vs2.29±0.36% uptake/1×105 cells, not significant, or 1.66±
0.22% uptake/1×105 cells, p<0.05; at 72-h incubation:
13.11±3.05% uptake/1×105 cells vs 4.24±1.13% uptake/
1×105 cells, p<0.001 or 2.31±0.42% uptake/1×105 cells,
p<0.001) (Fig. 2a). On the other hand, there was a marked
reduction of FLT uptake in the cells incubated in themedium containing DTX at 24 h of incubation (1.24±0.53%
uptake/1×105 cells at standard medium, 0.24±0.04% uptake/
1×105cells, p<0.001 at 10 nM DTX, 0.44±0.14% uptake/1×
105cells, p<0.05 at 100 nM DTX) (Fig. 2b).
Flow cytometric study

2.5

After 24-h incubation in each medium, G1, S and G2+M
fractions in the cells were determined. There was a marked
reduction of % S-phase cell fraction of the cells incubatedin the medium with DTX (28.45±1.39% at standard
medium, 17.80±1.36%, p<0.05 at 10 nM DTX; 15.33±
5.31%, p<0.01 at 100 nM DTX) (Fig. 2c).

2
1.5
1
0.5
0

24
4

48

72

Incubation time (h)Fig. 1 The number of 22Rv1 cells among the standard medium andthe media with 10 nM or 100 nM DTX (**p<0.01) (a) and the PSA
concentration in three groups (***p<0.001) (b)MicroPET study
Tumour-bearing mice treated with DTX (n=6) or vehicle(n=6) for 2 weeks were scanned with the MicroPET

Eur J Nucl Med Mol Imaging (2011) 38:81–89

a

18
16

%Uptake / 1x105 cells

85

14
12

0nm
10nm
100nm

10
8
6
4
2
0

24

48

72

Incubation time (h)b2.5%Uptake / 1x105 cells

0nm
10nm

2

100nm
1.5
1

Immunohistochemical examination of the 22Rv1 tumours
0.5
0

*
****
24

48

72

Incubation time (h)% S-phase cell fractioncwith no significant difference in the tumour growth rate
between DTX- and vehicle-treated animals (DTX vs vehicle:
2.98±2.25 vs 2.51±1.46) (Fig. 4a). Serum PSA value wasalso compared before and after 2-week DTX treatment,
showing no significant difference between two groups ofanimals (DTX vs vehicle: 3.90±3.41 vs 3.48±1.43) (Fig. 4b).
The FLT study results are shown in Fig. 4c. The tumour tomuscle ratios in the vehicle-treated mouse group were 5.34±
2.16 before treatment, and they were 3.79±1.28 after 2-week
vehicle treatment, indicating a slight but not significant
decrease in tracer uptake. On the other hand, the tumour tomuscle ratios in the DTX-treated mice were 8.72±3.67 before
treatment, and they were 2.82±0.53 after 2-week DTX
treatment, indicating a significant decrease of FLT uptake
after DTX treatment (p<0.05). The FDG study results areshown in Fig. 4d. There was no significant difference in FDG
uptake in the implanted tumour between the baseline value
and the value after DTX or vehicle treatment (baseline vspost-vehicle treatment: 3.32±1.34 vs 3.49±1.15; baseline vspost-DTX treatment: 4.22±1.28 vs 3.36±1.49).

35

**
*

30
25
20
15
10
5
0

0nM

10nM

The results of the cellular proliferative study using PCNA
staining are shown in Fig. 5a. The rates of cellular
proliferation in the vehicle- and DTX-treated mice were
9.82±3.12% and 3.49±0.53% after 2 weeks of treatment,
indicating a large decrease in the proliferative activity of theimplanted tumour following DTX treatment (p<0.05). The
results of an evaluation of the rate of apoptosis in thetumour using the TUNEL method are shown in Fig. 5b.
The apoptotic indices in the vehicle- and DTX-treated mice
were 0.78±0.12% and 0.67±0.12% after 2-week treatment,
indicating that there was no difference in the apoptotic
status between the two groups of mice.

100nM

Concentration of DTX (nM)
Fig. 2 FDG uptake in the cells among the three groups (*p<0.05,***p<0.001) (a), FLT uptake in the cells (***p<0.001, *p<0.05) (b)and % S-phase cell fraction (*p<0.05, **p<0.01) (c)scanner. The tumour size of each animal was also recorded.
MicroPET images using FLT or FDG clearly demonstrated
the tumours implanted in the left flank of the mice (Fig. 3).
Static images at 2 h post-injection for FLT and 1 h postinjection for FDG were reconstructed, and ROIs were placedon the lumbar muscle and tumour using the same transverse
images. Tracer uptake was compared in the same mice before
and after 2-week DTX treatment using tumour to muscle
ratios. Implanted tumours showed continuous growth during
treatment (DTX vs vehicle: 1,459.6±1,015.0 mm3 to 3,245.6±
2,096.7 mm3 vs 916.8±738.5 mm3 to 2,347.9±1,840.0 mm3),

Discussion
Monitoring the early therapeutic effects of chemotherapy,
including DTX therapy, is crucial for patients with HRPC,
because HRPC shows relatively rapid growth, leading topoor prognosis. Monitoring the anticancer effects isessential when deciding whether a certain kind of anticancer management should be maintained or should bechanged for a better clinical outcome. For this purpose,tumour imaging including CT or MRI and evaluating serum
PSA value are employed, which sometimes results in an
insufficient evaluation of the early therapeutic effects. At
this point, performing PET imaging of malignant tumours
has a tremendous advantage because PET can be used toassess the metabolic status of cancer before it changes its
anatomical form, which allows us to predict the reaction of

86

Eur J Nucl Med Mol Imaging (2011) 38:81–89

a

b

Tumor

Tumor

Tumor

Tumor

FLT PET week 0

FDG PET week 0

Fig. 3 MicroPET images of tumour implanted mice using FLT (a) and FDG (b)[21–24]. However, the short half-life of 11C (20 min) with
rapid degradation in vivo prevents 11C-thymidine fromseeing wide clinical use.
FLT is another thymidine analogue which has a longer
half-life (110 min) because it is labelled with 18F. According to a study by Shields et al., the FLT uptake in tissues
depends on their proliferation activity [15]. Rasey et al.
reported on the mechanisms of FLT uptake in the cell,showing that FLT uptake is positively correlated with cell

a

b

5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

Seeum PSA value (ng/mL)

Tumour growth rate

a tumour to the chemotherapeutic agents as early as
possible. Malignant tumours are characterized as tumours
with a deregulated cell cycle progression resulting in rapid
tumour growth. Enhanced proliferation of cancer cells
demands an increased supply of DNA substrates, which
are deoxynucleotides (dNTPs). Accordingly, analysis of
DNA metabolism is as important a strategy as cancer
imaging with PET. 11C-Thymidine was the first radiopharmaceutical to assess cell proliferation for in vivo imaging

8
7
6
5
4
3
2
1
0

VehicleVehicleDocetaxel
Treatment

12

Tumour/Muscle ratios*

14
pre-Txpost-Txd

14
12

Tumour/Muscle ratioscDocetaxel
Treatment

10
8
6
4
2

pre-Txpost-Tx

10
8
6
4
2

0

0
VehicleDTXTreatment

Fig. 4 Difference of tumour growth rate between the DTX- andvehicle-treated animals (a), difference of serum PSA value between
the DTX- and vehicle-treated animals (b), difference of tumour toVehicleDTXTreatment

muscle ratios of FLT uptake between the two groups (*p<0.05) (c)and difference of tumour to muscle ratios of FDG uptake between thetwo groups (d)Eur J Nucl Med Mol Imaging (2011) 38:81–89

Fig. 5 The rates of cellular proliferation in the vehicle- and DTXtreated mice (*p<0.05) (a) and apoptotic indices in the vehicle- andDTX-treated mice (b)growth and thymidine kinase 1 (TK1) activity and that
inhibition of the cell cycle progression prevents FLT uptake
and increased TK1 activity [25]. They concluded that FLT
images reflect TK1 activity and the percentage of cells in S
phase. These studies encouraged us to perform the current
study to determine whether FLT is a useful tracer formonitoring the early therapeutic effects of DTX on HRPCin vitro and in vivo and also to determine whether FLT
uptake in tumours correlates with actual proliferative
activity after DTX therapy.
In our current study, DTX induced a significant
inhibition of cell growth at 48 or 72 h post-treatment but
not at 24 h in vitro. Production of PSA by the prostatecancer cells showed only a minimum inhibition at 24 h.
These data indicate that 24-h DTX treatment is not longenough to induce an anticancer effect on tumour growth or
the PSA production of 22Rv1 cells in vitro. Thus, longer
treatment is needed to reveal such a difference. However,the flow cytometric study of the cells showed that there wasa decrease in the S-phase cell fraction at 24 h of DTX
treatment. This clearly shows that there is a chance tovisualize the change in this DNA synthesis if we employ anadequate radiotracer to monitor the activity of DNA
synthesis of the cells. We analysed two state-of-the-art
PET tracers, FDG and FLT, for this purpose. As shown inthis study, there was only a small change in FDG uptake in87

the 22Rv1 cells at 24 h of DTX treatment, which showedthat the cells in the early phase of cell cycle arrest may havenormal glucose metabolism. This result agrees with theprevious finding of an insignificant change in FDG uptake
after chemotherapy [26]. On the other hand, there was amarked reduction of FLT uptake in the cells at 24 h of DTX
treatment, whereas FDG uptake was still unchanged at thistime point. This result clearly showed that FLT is apromising tracer for monitoring the early therapeutic effectof DTX on prostate cancer cells based on the changes of
DNA synthesis. This result agrees with another study
reporting a low uptake of FLT in cells treated with
anticancer drugs [25].
In our animal study, we succeeded in visualizing implanted
22Rv1 tumours using microPET with FLT. We calculated thetumour growth rates of the implanted tumour by measuring
the actual tumour size before and after 2-week treatment withvehicle or DTX and found that there was only a minimal
difference in tumour size between before and after treatment
with either vehicle or DTX. There was also no significant
difference in the post-treatment change in the apoptotic index
of the tumour between the two treatment groups. This
indicated that 2-week DTX treatment may not be sufficient
to induce anatomical changes of the implanted tumour even if
animal CT is employed. Measuring serum PSA was not useful
to monitor the anti-tumour effect of DTX at 2 weeks after
treatment. These data indicate that 2-week treatment with
DTX did not fully affect the tumour volume or PSA
production in the tumour, and thus it seems to be difficult toobserve the effects of DTX on HRPC using conventional
monitoring methods. However, the immunohistochemicalevaluation using the PCNA method demonstrated that there
was a decrease in proliferative cells in the tumour implanted
into the animals treated with DTX. This clearly showed thatthe evaluation of DNA synthesis in tumour cells can be an
ideal strategy to determine the early anticancer effects ofchemotherapeutic agents.
In the microPET study, a significant decrease of FLT uptake
in tumours was demonstrated following 2-week DTX treatment, while there were no significant changes in the vehicle
group. On the other hand, FDG uptake in tumours showed
only minimal change in both groups. These microPET study
results were well correlated with a change in the proliferation
activity of the tumour determined by PCNA staining. This
may indicate that 2-week DTX treatment of these animals
influenced the TK1 activity of 22Rv1 cells, which has been
well evaluated with FLT uptake. It is presumed that thischange of TK1 activity comes into existence earlier than thatof glucose metabolism, which can be evaluated with FDG
uptake. Thus, FLT PET is superior to FDG for monitoringtumour proliferation activity after chemotherapy.
Our in vitro and in vivo studies both indicate that DTX
treatment for a short period of time results in cell arrest of

88

22Rv1 prostate tumour cells without enhanced apoptotic
cell death, and without reduction of both PSA production
and glucose metabolism, while FLT PET appears to be
useful as a tumour proliferation marker after DTX therapy.
Ebenhan et al. recently reported the suitability of FDG,
FLT, 18F-methionine and 18F-fluorocholine as noninvasive
PET biomarkers for monitoring the response to chemotherapy [27]. In another study, they monitored the effects ofpatupilone, a novel cytotoxic compound that is a potent
microtubule stabilizer [28], on FLT uptake in RIF-1 tumours
and observed a significant decrease of tracer uptake after
24 h, which showed a strong negative correlation to apoptosis.
Their results were consistent with our current study.
FLT has been well known for its high uptake to bonemarrow. This feature of FLT may be helpful in evaluation of
the effects of anticancer therapy on bone marrow for HRPC.
According to the clinical study for oesophageal carcinoma by
Yue et al., marked early reduction of proliferation in irradiatedmarrow was observed after only 2 Gy and complete absenceof proliferation after 10 Gy [29]. They concluded that FLT
PET can depict the effects of radiotherapy on bone marrow.
Metastatic bone lesions are one of the common targets for
DTX treatment for patients with HRPC. FLT PET could havea potential to evaluate the early anticancer effect of DTX on
HRPC. Further in vivo and clinical investigations are needed
to determine the role of FLT PET for bone marrow lesions.
However, tracer uptake in bone marrow could be a limitationof FLT PET imaging for patients with HRPC when screening
small osseous metastatic lesions. Detailed clinical study
using FLT PET for patients with HRPC is needed.
One of the limitations of the current study was that many
important parameters for androgen-independent prostatecancer such as serum androgen levels were neglected. A
majority of the patients who have androgen-independent
prostate cancer receiving chemotherapy including docetaxel
also receive anti-androgen therapy, which lowers serum
testosterone levels. This may also affect behaviour of FLT
uptake in prostate cancer tissues. Further in vivo investigation seems to be necessary to clarify the role of FLT PET
imaging for those patients receiving both DTX and AAT.
These results indicate that FLT is a promising tracer formonitoring the early therapeutic effects of DTX in patientswith HRPC. Thus, FLT PET has potential for visualizing
early-phase changes in the proliferation activity of HRPC
after DTX treatment. Our data also suggest that FLT issuperior to FDG in evaluation of the early therapeutic effectof DTX on HRPC.

Conclusion
These results indicate that 18F-FLT is a promising tracer formonitoring the early therapeutic effects of DTX on HRPC.

Eur J Nucl Med Mol Imaging (2011) 38:81–89

Further clinical studies in humans will be needed toevaluate the possibility of using FLT PET to monitor the
early therapeutic effects of DTX therapy.
Acknowledgements This study was partially supported by a 21stCentury COE Grant (Medical Science) and by a Grant-in-Aid for
Scientific Research (C) from the Japan Society for the Promotion of
Science. This study was also supported through the Washington
University Small Animal Imaging Resource Grant (U24 CA083060).
The NCI Cancer Center Support Grant (P30 CA91842) supports the
Small Animal Imaging Core as the Alvin J. Siteman Cancer Center,
Washington University and Barnes-Jewish Hospital in St. Louis. The
authors wish to thank Lori A. Strong, Jerrel R. Rutlin, Nicole M.
Fettig and Terry L. Sharp for their technical support with themicroPET imaging and Reiko Oyama and Sally W. Schwarz for the
production of isotopes and helpful discussion. A preliminary report ofthis work was presented at the 52th Annual Meeting of the Society of
Nuclear Medicine in Toronto in 2005.
Conflicts of interest None.

References
1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer
statistics, 2009. CA Cancer J Clin 2009;59:225–49.
2. Huggins C, Hodges CV. Studies on prostatic cancer: I. The effectof castration, of estrogen and of androgen injection on serum
phosphatases in metastatic carcinoma of the prostate. Cancer Res
1941;1:293–7.
3. Eisenberger MA. Chemotherapy for prostate carcinoma. NCI
Monogr 1988;7:151–63.
4. Petrylak DP, Tangen CM, Hussain MH, Lara PN Jr, Jones JA,
Taplin ME, et al. Docetaxel and estramustine compared withmitoxantrone and prednisone for advanced refractory prostatecancer. N Engl J Med 2004;351:1513–20.
5. Oyama N, Kim J, Jones LA, Mercer NM, Engelbach JA, Sharp
TL, et al. MicroPET assessment of androgenic control of glucose
and acetate uptake in the rat prostate and a prostate cancer tumor
model. Nucl Med Biol 2002;29:783–90.
6. Oyama N, Akino H, Suzuki Y, Kanamaru H, Ishida H, Tanase K,
et al. FDG PET for evaluating the change of glucose metabolism
in prostate cancer after androgen ablation. Nucl Med Commun
2001;22:963–9.
7. Kurdziel KA, Figg WD, Carrasquillo JA, Huebsch S, Whatley M,
Sellers D, et al. Using positron emission tomography 2-deoxy-2[18F]fluoro-D-glucose, 11CO, and 15O-water for monitoring
androgen independent prostate cancer. Mol Imaging Biol
2003;5:86–93.
8. Effert PJ, Bares R, Handt S, Wolff JM, Büll D, Jakse G. Metabolic
imaging of untreated prostate cancer by positron emission
tomography with 18fluorine-labeled deoxyglucose. J Urol 1996;
155:994–8.
9. Oyama N, Akino H, Suzuki Y, Kanamaru H, Sadato N, YonekuraY, et al. The increased accumulation of [18F]fluorodeoxyglucose
in untreated prostate cancer. Jpn J Clin Oncol 1999;29:623–9.
10. Hara T, Kosaka N, Kishi H. PET imaging of prostate cancer using
carbon-11-choline. J Nucl Med 1998;39:990–5.
11. DeGrado TR, Baldwin SW, Wang S, Orr MD, Liao RP, Friedman
HS, et al. Synthesis and evaluation of (18)F-labeled choline
analogs as oncologic PET tracers. J Nucl Med 2001;42:1805–14.
12. Oyama N, Akino H, Kanamaru H, Suzuki Y, Muramoto S,
Yonekura Y, et al. 11C-acetate PET imaging of prostate cancer. J
Nucl Med 2002;43:181–6.

Eur J Nucl Med Mol Imaging (2011) 38:81–89
13. Yoshimoto M, Waki A, Yonekura Y, Sadato N, Murata T, Omata
N, et al. Characterization of acetate metabolism in tumor cells in
relation to cell proliferation: acetate metabolism in tumor cells.
Nucl Med Biol 2001;28:117–22.
14. Yoshimoto M, Waki A, Obata A, Furukawa T, Yonekura Y,
Fujibayashi Y. Radiolabeled choline as a proliferation marker:
comparison with radiolabeled acetate. Nucl Med Biol 2004;31:
859–65.
15. Shields AF, Grierson JR, Dohmen BM, Machulla HJ, Stayanoff JC,
Lawhorn-Crews JM, et al. Imaging proliferation in vivo with [F-18]
FLT and positron emission tomography. Nat Med 1998;4:1334–6.
16. Buck AK, Schirrmeister H, Hetzel M, Von Der Heide M, Halter
G, Glatting G, et al. 3-Deoxy-3-[18F]fluorothymidine-positronemission tomography for noninvasive assessment of proliferationin pulmonary nodules. Cancer Res 2002;62:3331–4.
17. Vesselle H, Grierson J, Muzi M, Pugsley JM, Schmidt RA,
Rabinowitz P, et al. In vivo validation of 3'deoxy-3'-[(18)F]
fluorothymidine ([(18)F]FLT) as a proliferation imaging tracer inhumans: correlation of [(18)F]FLT uptake by positron emission
tomography with Ki-67 immunohistochemistry and flow cytometry
in human lung tumors. Clin Cancer Res 2002;8:3315–23.
18. Pio BS, Park CK, Pietras R, Hsueh WA, Satyamurthy N, Pegram
MD, et al. Usefulness of 3'-[F-18]fluoro-3'-deoxythymidine withpositron emission tomography in predicting breast cancer
response to therapy. Mol Imaging Biol 2006;8:36–42.
19. Ponde DE, Oyama N, Welch MJ, McCarthy TJ. Synthesis offluorothymidine ([18F]FLT) for radiotracer of tumors. Abstr Pap
AM Chem S 2002;223:081-Nucl Part 2.
20. Tai YC, Chatziioannou A, Siegel S, Young J, Newport D, Goble RN,
et al. Performance evaluation of the microPET P4: a PET system
dedicated to animal imaging. Phys Med Biol 2001;46:1845–62.

89
21. Shields AF, Lim K, Grierson J, Link J, Krohn KA. Utilization oflabeled thymidine in DNA synthesis: studies for PET. J Nucl Med
1990;31:337–42.
22. Goethals P, van Eijkeren M, Lodewyck W, Dams R. Measurementof [methyl-carbon-11]thymidine and its metabolites in head andneck tumors. J Nucl Med 1995;36:880–2.
23. Shields AF, Mankoff DA, Link JM, Graham MM, Eary JF,
Kozawa SM, et al. Carbon-11-thymidine and FDG to measuretherapy response. J Nucl Med 1998;39:1757–62.
24. Wells JM, Mankoff DA, Muzi M, O’Sullivan F, Eary JF, Spence
AM, et al. Kinetic analysis of 2-[11C]thymidine PET imaging
studies of malignant brain tumors: compartmental model investigation and mathematical analysis. Mol Imaging 2002;1:151–9.
25. Rasey JS, Grierson JR, Wiens LW, Kolb PD, Schwartz JL.
Validation of FLT uptake as a measure of thymidine kinase-1
activity in A549 carcinoma cells. J Nucl Med 2002;43:1210–7.
26. Dittmann H, Dohmen BM, Kehlbach R, Bartusek G, Pritzkow M,
Sarbia M, et al. Early changes in [18F]FLT uptake after chemotherapy: an experimental study. Eur J Nucl Med Mol Imaging
2002;29:1462–9.
27. Ebenhan T, Honer M, Ametamey SM, Schubiger PA, Becquet M,
Ferretti S, et al. Comparison of [18F]-tracers in various experimental tumor models by PET imaging and identification of anearly response biomarker for the novel microtubule stabilizer
patupilone. Mol Imaging Biol 2009;11:308–21.
28. Altmann KH. Epothilone B and its analogs – a new family of
anticancer agents. Mini Rev Med Chem 2003;3:149–58.
29. Yue J, Chen L, Cabrera AR, Sun X, Zhao S, Zheng F, et al.
Measuring tumor cell proliferation with 18F-FLT PET during
radiotherapy of esophageal squamous cell carcinoma: a pilot
clinical study. J Nucl Med 2010;51:528–34.